Optical traps, or sometimes referred to as optical tweezers, utilize a light source to produce radiation pressure. Radiation pressure is a property of light that creates small forces by absorption, reflection, or refraction of light by a dielectric material. Relative to other types of forces, the forces generated by radiation pressure are almost negligible--only a few picoNewtons (1 pN=1.times.10.sup.-12 N) from a light source of a few milliwatts of power. However, a force of a few picoNewtons is more than sufficient to represent the interactions of microscopic molecules such as actin and myosin in the presence of adenosine triphosphate (ATP).
The present invention utilizes the gradient force that exists when a transparent material with a refractive index greater than the surrounding medium is placed in a light gradient. As light passes through polarizable material, it induces a dipole moment. This dipole interacts with the electromagnetic field gradient, resulting in a force directed towards the brighter region of the light, normally the focal region. However, if an object has a refractive index lower than the surrounding medium, such as an air bubble in water, the object experiences a force drawing it toward the darker region. Optical traps utilize brighter regions or focal points of light to draw the specimen toward the direction of the focal region of the light source.
As long as the frequency of the laser is below the natural resonances of the particle being trapped (e.g., the absorption edge of a polystyrene sphere), the dipole moment is in phase with the driving electric field. The energy of the particle can be described as EQU W=-p.multidot.E
where,
W=energy of the induced dipole in the electric field
p=induced dipole moment of the particle
E=electric field
Thus, the particle minimizes its energy by moving to the region where the electric field is the highest, namely the focal point of the laser beam.
A simplified model of the optical trap is as follows: light, such as laser light, enters a high numerical aperture objective lens of an optical system and is focused to a diffraction-limited region or spot on a spherical object in the specimen plane. Because the intensity profile of the laser light is not uniform, an imbalance in the reaction forces generates a three-dimensional gradient force with the brightest light in the center. The gradient force pulls the object toward the brighter side. Thus, the picoNewton forces generated by the optical system "traps" the object. Such gradient forces are formed near any light focal region.
The sharper or smaller the focal region, the steeper the gradient. To overcome scattering forces near the focal region and hence prevent the object from being ejected along the direction of the light beam, the optical system must produce the steepest possible gradient forces. Sufficiently steep gradient forces can be achieved by focusing laser light to a diffraction-limited spot of diameter of approximately .lambda., the laser light wavelength, through a microscope objective of high numerical aperture (N.A.). Appropriately enough, this single-beam gradient force optical trap is also known as "optical tweezers."
The magnitude of forces produced by this optical system is on the scale of picoNewtons. These lower magnitude forces are the kinds of forces encountered at the biological ultrastructure level. PicoNewton size forces can move cells, bend cell elements, impede organelle or bacteria movements, and overcome the motion of biological motors, such as myosin and kinesin.
One embodiment of the present invention utilizes optical traps to study motor molecules, where the forces generated by these mechanoenzymes are in the picoNewton range. Micrometer-sized spheres called "handles" can be attached to a sample such as an actin filament. These micrometer-sized handles can be, for example, refractile silica or latex spheres. These handles are optically trappable by a focused laser light source. Additionally, their symmetry and uniform content facilitate calibration against Stokes' drag.
Note that these handles attached to the sample are a narrow class of particles that can be optically trapped. In fact, the present invention, through its embodiments, can be used with any opticaly trappable particle. Any neutral particle that can be manipulated by the small scale negative radiation pressure formed by a focal region of a light source can be used. The particle can be, among other things, an atom, a dielectric particle whose size is in the range of 10 .mu.m to approximately 25 nm, a Mie particle, and a Rayleigh particle.
The particles trapped by the focal point of the light, and thus the attached sample, can be steered with lenses, galvanometer mirrors, and other optical devices. Multiple optical traps can also be utilized for various assay purposes such as spatial orientation and pulling taut a sample. Multiple optical traps expand upon the utility of single optical traps.
Prior optical traps had little or no stiffness; that is, the particles trapped by the optical trap did not maintain their desired on-target position. Furthermore, these multiple optical traps did not use feedback signals to stably hold the position of the particles in both the x and y directions in the sample region. With feedback, the optical trap system can be used to stiffen the particle's position for greater flexibility in particle manipulation. In one of many applications, an optical trap system with feedback can be used to study and measure the interaction and forces of the surrounding protein motor molecules, such as myosin, with the "handled" molecule, such as actin filament. The level of feedback signal required to close the loop and hold the molecules in a stable location also provides a measure of the force generated by the trapped object. Accordingly, an embodiment of the present invention can measure the force produced by a single myosin molecule as they move against a single actin filament in the presence of varying concentrations of ATP. Two traps with feedback position control are used so that a flexible actin filament can be manipulated and stretched taut between two handles in space.
During muscle contraction chemical energy from ATP hydrolysis is converted to relative sliding of actin and myosin filaments to produce force. Although the actomyosin system has been extensively studied, the mechanism underlying its mechanochemical energy action remains unknown. The conventional swinging crossbridge theory suggests that for each ATP hydrolysis, myosin binds to actin and undergoes a conformational change or power stroke before subsequently detaching. This theory assumes that the myosin step size, or movement of actin relative to myosin for each ATP hydrolysis, is less than 40 nm, a value limited by the physical dimensions of the myosin head. Recently, various studies showed step sizes that were inconsistent with the conventional theory. To resolve this issue, the present invention provides a new technique with the resolution to probe the mechanical properties of myosin at the level of single molecular events.
The optical trap technique disclosed herein can measure single molecular events, such as displacement and force, and offers advantages over other force measuring techniques such as the use of microneedles. The advantages include ease of use and, with feedback positional control, the ability to change the stiffness of the trap in the middle of an experiment. In one experiment conducted with an embodiment of the present invention, the optical trap with feedback in motility assays allowed the measurement of nanometer movements and picoNewton forces at millisecond rates of samples on a coverglass. By adding a second optical trap an actin filament can be held and manipulated via beads, or handles, attached to each end of the actin filament which prevents the actin filament from diffusing away from surfaces sparsely coated with myosin. By placing myosin molecules on a bead support above the coverglass surface, interactions of either the actin filament or the handles with the microscope coverslip surface are minimized. An electronic feedback system allowed the measurement of forces under approximately isometric conditions.